The present invention relates generally to biotechnology, and in particular to novel uses and production of Growth Hormone Releasing Hormone Related Peptide (GHRH-RP).
As further background, Growth Hormone Releasing Hormone (GHRH) is known to be the hypothalamic factor that stimulates the release of pituitary growth hormone. Rat GHRH mRNA and peptide have been identified in multiple tissues outside of the hypothalamus including gut, placenta and gonads. M. O. Thorner et al., Acta Endocrinol [Suppl] (Copenh) 276, 34 (1986); T. O. Bruhn, R. T. Mason, W. Vale, Endocrinology 117, 1710 (1985); O. H. Pescovitz, N. Johnson, S. A. Berry, Pediatr. Res. 29, 510 (1990); G. Meigan, A. Sasaki, K. Yoshinaga, Endocrinology 123, 1098 (1988); A. Bagnato, C. Moretti, J. Ohnishi, G. Frajese, K. J. Catt, Endocrinology 130, 1097 (1992); S. A. Berry and O. H. Pescovitz, Endocrinology 123, 661 (1988). Southern blot analysis of rat genomic DNA indicates that there is a single GHRH gene. Alternate first exons are used to regulate differential GHRH mRNA transcription in various tissues. K. E. Mayo, G. M. Cerelli, M. G. Rosenfeld, R. M. Evans, Nature 314, 464 (1985); Gonzalez-Crespo and A. Boronat, Proc. Natl. Acad. Sci. USA 88, 8749 (1991); C. H. Srivastava, B. S. Monts, J. K. Rothrock, M. J. Peredo, O. H. Pescovitz, Endocrinology 136, 1502 (1995). Exons 2 through 5 are common to all tissues and encode for a 104 amino acid GHRH precursor peptide. This peptide is cleaved into a 29 amino acid N-terminal signal peptide, the mature 43 amino acid GHRH peptide, and a putative 30 amino acid C-terminal peptide, referred to as GHRH-Related Peptide (GHRH-RP).
GHRH is postulated to have arisen from a single ancestral gene approximately 1250 million years ago. R. M. Campbell and C. G. Scanes, Growth Regulation 2, 175 (1992). This gene is believed to give rise to the various GHRH family members including glucagon, secretin, pituitary adenylate cyclase activating peptide (PACAP), and vasoactive intestinal peptide (VIP). While the other members of this family are known to produce more than one functional peptide from their precursor proteins, exon loss was hypothesized to explain the occurrence of a single peptide product for both the GHRH and secretin genes. R. M. Campbell and C. G. Scanes, Growth Regulation 2, 175 (1992). Pre-pro-VIP produces both peptide histidine isoleucine and VIP and proteolytic cleavage of the PACAP precursor produces PACAP-related peptide and PACAP.
The GHRH mRNA and peptide and other members of the GHRH family have previously been identified in testicular tissue. S. A. Bery, C. H. Srivastava, L. R. Rubin, W. R. Phipps, O. H. Pescovitz, J. Clin. Endo. Metab. 75, 281 (1992); M. Ohta, S. Funakoshi, T. Kawasaki, N. Itoh, Biochem. Biophys. Res. Comm. 183, 390 (1992); S. Shioda et al., Endocrinology 135, 818 (1994); R. Hakanson, F. Sundler, R. Uddman, in Vasoactive Intestinal Peptide, S. Said, Ed. (Raven Press, New York 1982), pp. 121-144. The GHRH transcript is localized to spermatogenic cells, is developmentally regulated and is actively transcribed during the onset of spermatogenesis. C. H. Srivastava et al., Endocrinology 133, 83 (1993); S. A. Berry and O. H. Pescovitz, Endocrinology 127, 1404 (1990). Northern blot analysis of RNA from separated testicular cells demonstrates the highest amounts of GHRH mRNA in spermatocytes and immature round spermatids. GHRH mRNA is not present in more mature elongating spermatids and epididymal sperm. Using immunohistochemical analysis, the GHRH peptide is localized to both germ cells (O. H. Pescovitz et al., Endocrinology 127, 2336 (1990)) and Leydig cells (T. Ciampani, A. Fabbri, A. Isidori, M. L. Dufau, Endocrinology 131, 2785 (1992); A. Fabbri, D. R. Ciocca, T. Ciampani, J. Wang, M. L. Dufau, Endocrinology 136, 2303 (1995)). A GHRH receptor is transcribed in rat Sertoli cells (C. H. Srivastava et al., Endocrine Journal. 2, 607 (1994)) and treatment of cultured Sertoli cells with GHRH activates Sertoli cell c-fos and stem cell factor gene expression (C. H. Srivastava, P. R. Breyer, J. K. Rothrock, M. J. Peredo, O. H. Pescovitz, Endocrinology 133, 1478 (1993)). Both of these Sertoli cell products are crucial for the normal progression of spermatogenesis. R. S. Johnson, B. M. Spiegelman, V. Papaioannou, Cell 71, 577 (1992); K. M. Zsebo et al., Cell 63, 213 (1990); E. Huang et al., Cell 63, 225.
In the area of fertility management, the literature has taught the treatment of human female infertility by administration of gonadotropins such as follicle stimulating hormone (FSH) combined with luteinising hormone (LH), and the the addition of GHRH to this mixture to improve the therapy. Tests have also been derived to predict whether the FSH/LH/GHRH regimen will benefit the patient. See, e.g., U.S. Pat. No. 5,175,111. In this area it is also known that inhibition of FSH, for example with FHS-Inhibiting Protein (FSH-IP), can be used to provide contraception in males and females, since FSH is required for maturation of ovarian follicles and testicular spermatogenesis. In addition, disruption of FSH-IP activity, e.g. by administration of antibodies to FSH-IP, has been taught as a means for promoting fertility. See, e.g., U.S. Pat. No. 5,037,805.